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What is the definition of muscular dystrophies?

- group of inherited disorders of muscle in which muscle histology has certain distinctive features (muscle fibre necrosis, phagocytosis etc) where there is no clinical or laboratory evidence of central or peripheral nervous system involvement or myotonia


What is the most severe and most common muscular dystrophy?

- duchenne muscular dystrophy


What are symptoms of DMD?

- floppy muscles (hypotonia) - sometimes
- sometimes delay in walking, toe walking
- clumsy, falling over, can't run properly (waddling gait)
- gower sign ('climbing up' legs from lying position)
- muscle pseudohypertrophy (excess fat and connective tissue)
- lumbar lordosis (sway back) and protuberant abdomen
- IQ < 75 in ~ 30% of cases, speech delay


What is pseudohypertrophy?

- starts out true hypertrophy - muscle cells get larger
- over time skeletal muscle tissue is destroyed and the tissue is replaced by connective tissue and fat
- hallmark feature of DMD


What is the cause of symptoms in DMD?

- increasing proximal muscle weakness due to progressive muscle degeneration
- caused by mutation(s) in a gene encoding a muscle protein called dystrophin
- dystrophin found in all muscle (sub sarcolemma) and brain
- forms link between actin (cytoskeletal) and extracellular matrix
- many isoforms of the protein that have different promoters that regulate gene expression in different tissue types


What is the dystrophin-associated protein complex (DAPC)?

- dystrophin is part of an intracellular-transmembrane-extracellular complex
- dystrophin forms rod like structure underneath the sarcolemma
- dystrophin forms internal link with actin at N-terminal end
- interactions at C-terminal
- interactions across its body with a number of proteins in the plasma membrane that link to laminin


What are the effects of dystrophin deficiency?

1. absence of/or altered dystrophin
2. membrane instability
3. increased Ca2+ influx
4. increased proteolytic and lipolytic enzyme activity
5. muscle degradation
6. muscle regeneration

muscle degradation > muscle regeneration --> DMD/BMD


What are consequences in DMD?

- spinal curve to one side
- may required surgery to insert metal rod

wheelchair usually by 12 years of age

respiratory/cardiac failure

death: late teens-20s (av 19 years)
can now be into 30s


What are current treatments?

medical (steroids), occupational therapy, physiotherapy
improve outcomes


Compare duchenne and becker muscular dystrophies

- more severe
- wheelchair by 12 years
- zero fertility (fitness = 0)

- milder (adults survive)
- may never have wheelchair; never before 16
- fertility reduced (fitness = 0.7)

both conditions now known to be due to different types of mutations in the same gene (allelic variants)


What is the inheritance of DMD?

- X-linked recessive
- in 2/3 of isolated cases, mother is a carrier (DMD)
- mutations can occur de novo
- 15 - 45% of carriers have mild symptoms - skewed X-inactivation (called manifesting heterozygotes)
- ~1/3 due to spontaneous (de novo) mutations
-- gonadal mosaics in about 6%
-- new mutations in eggs or early developing embryo

can be complicated working out if the mother is a carrier or not


What is the prevalence of DMD/BMD?

of liveborn male births:
DMD: 1/3,500
BMD: ~ 1/20,000


What is dystrophin gene and protein?

- Xp21
- 2.5Mb
- 79 exons
- 7 distinct promoters
- 14kb mRNA
- gene product 427 kDa

- rod like protein:
-- actin-binding domain
-- rod domain (repetitive domains)
> could survive/have milder symptoms if fewer rod domains
-- cysteine-rich domain
-- c-terminal domain

- tissue specific isoforms, under different promoters

- second largest gene after titan

- multiple promoters
- e.g. C1, M1, P1, R1, CNS1, S1, G1
- cortex brain, muscle, purkinje, retinal (+ brain + cardiac muscle); CNS, central nervous system (+ kidney); S (Schwann cell), G general


What is the importance of reading frame?

- becker is milder because there is some protein reduced (even if mutant) and this results in milder phenotype
- 2/3 of mutations in duchenne are deletions of either 1 or more exons
- these deletions disrupt the reading frame

- duchenne mutations disrupt reading frame and cause premature termination of protein and non functioning protein - mRNA usually degraded
- out-of-frame deletion
- other mutations that are severe enough they result in no protein etc --> duchenne

- becker mutations cause reading frame to be intact
- a short but partially functional protein results


How are DMD and BMD diagnosed?

- creatine kinase (blood) - a screening test
- pathology of muscle biopsy ('gold' standard - but now only done if DNA testing does not provide a result)
- DNA tests
-- direct testing (deletions); sequencing
-- indirect testing (linkage)


What is creatine kinase as a diagnostic?

- muscle isoform of creatine kinase levels are elevated in serum (20 to 50x)
normal range: 40 - 240 U/L
e.g. DMD: 12028 U/L


What is the pathology of muscle biopsy (DMD)?

- abnormal variation in diameter of the muscle (atrophy and hypertrophy), central nuclei
- focal necrotic and regenerative fibres
- foci of inflammatory cells
- extensive replacement of muscle fibres with fat and fibrous connective tissue
- immunocytochemistry - <5% dystrophin positive fibres (using immunoperoxidase)

normal: regular size cells, normal vasculature
DMD: different sizes, increased connective tissue and fat, some cells hyper stain, some very pale, nuclei move more centralised, foci inflammatory cells, disorganised architecture
- no dystrophin seen


What is BMD muscle pathology?

- immunocytochemistry - dystrophin normal or reduced
- milder pathologically


How would we do DNA testing to diagnose duchenne/BMD?

mainly deletions (65% of DMD, 85% of BMD)
- 100% of deletions detected by 3 multiplex PCRs (A, B, C for 24 exons most commonly deleted)
- primer pairs to amplify 6 (or more) exons used in a single reaction
- difficult to detect 1/3 of point mutations in DMD gene
- this direct DNA test is not quantitative and has now been superseded


What is MLPA?

- more recent
- multiplex ligation-dependent probe amplification - MLPA
-- PCR based
- can quantitatively detect deletions or duplications in all 79 exons of dystrophin gene
-- can detect carriers of deletions or duplications – DMD and BMD
- probes designed so that only detected if can hybridise to corresponding exon
- limitation:
-- does not detect the remaining 1/3rd of cases due to point mutations - sequencing ??
-- microdeletions or point mutations around ligation site will be reported as deletions

features of MLPA probes
- each one specific for the exon to be tested
- universal primer
- target sequence A: 20-30 nucleotides complementary to sequences on exon
- target sequence B: 25 - 25 nucleotides complementary to sequences on exon
- stuffer fragment between 19 and 370 bp --> gives a different size product to distinguish between 79 exons
- universal primer


What are the five steps for MLPA testing?

1. denature DNA
2. hybridise proteins
3. ligate probes
4. PCR amplification
5. detection and analysis


What is the MLPA result for carrier deletion in DMD?

peaks for each exon e.g.
- note the peaks for exons 46 and 47 are half the size in the patient sample compared with the control sample, indicating that she has a deletion in these two exons on one of her X chromosomes


What is linkage analysis?

- linkage analysis is done when deletions are not found in the boy and there is a need to determine the disease-causing chromosome in the family
-- i.e. no deletion in previous affected family member
-- used for carrier testing
-- or for prenatal diagnosis

- linkage involves tracking the mutated dystrophin allele associated with the disease through the family - often uses CA repeats (polymorphic short tandem repeats, STRs) in introns within the dystrophin gene
-- NB recombination (crossing over) at meiosis can confound result

e.g. polymorphism of the STR49 marker
STR49: this marker represents dinucleotide CA repeats in intron 49 of the DMD gene
- this marker is polymorphic i.e. there are multiple alleles of STR49 with varying lengths found in the population
- these are X chromosomes in four individuals
-- persons A, B, and C are females
-- person D male

- the particular STR49 alleles are indicated below each chromosome as a line (i.e. the line represents the chromosome) with a number, which represents the product size (in base pairs) following amplification by PCR


What is carrier testing?

- knowing carrier status informs reproductive risk for future children
- if mutation in boy has been identified then do MLPA on at-risk relatives (mother etc) to determine carrier status
- if mutation in boy is not detected by MLPA, then can calculate (using computer program) final likelihood of being a carrier by combining
-- pedigree
-- linkage result (% recombination risk included)
-- and CK results (2/3 of all carriers have raised CK levels)


What are future therapies for DMD?

- gene therapy - may need to use modified 'mini-gene' because dystrophin gene so large

- up-regulation of alternative proteins e.g. utrophin (dystrophin homologue, higher levels in foetal tissue)

- anti-sense oligonucleotides that allow 'skipping' of exons to produce shorter partial functioning protein
-- Drisapersen for exon 51; PRO044 for exon 44, PRO045 for exon 45

- drugs to restore dystrophin production in the 10-15% of cases of DMD due to 'stop/nonsense' mutations (allow 'read through')
-- ataluren (PTC124), Gentamycin

- drugs to inhibit protein degradation or increase muscle mass, strength or function
-- e.g. flavocoxid, pentoxifylline

- myoblast transfer therapy - myoblasts containing normal dystrophin gene are implanted

- stem cell therapy - stem cells containing the normal gene for dystrophin are implanted

- hope for cure/prevention